Manufacture of dihydrofollicle hormones by fermentative reduction



' atente Dec, 19, 1939 lTED STATES or DIHYDROFOLLICLE MANUFACTUREHORMONES BY FERMENTATIVE REDUC- TION Walter Schoeller,-Berlin-Charlottenburg, Germany, assignor to ScheringCorporatiomBloomfield, N. .11., a corporation of New Jersey No Drawing.Application February 12, 1938, Se-

rial No. 190,226. 1937 '9 Claims.

This invention relates to a process for the manufacture ofdihydrofollicle hormones and analogues of the same containing lesshydrogen, as well as their derivatives, such as esters, ethers and thelike and is an improvement in or modification of the inventions of US.applications Ser. No. 694,686 filed October 21, 1933, now

Patent No. 2,096,744, issued October 26, 1937, and

Ser. No. 52,084 filed November 29, 1935, now Patent No. 2,072,830,issued March 2, 1937.

The said specifications describe the reduction of the keto group of thefollicle hormone, or analogues of the same containing less hydrogen,such as equilin, or its derivatives, such as esters,

l5 ethers and the like by means of chemical methods, including catalyticmethods and the use of nascent hydrogen, whereby a mixture of isomericdihydrofollicle hormones, or analogues of the same containing lesshydrogen, or their dem rivatives is produced. By the application ofthese chemical methods there is produced not only a mixture of isomerswhich possess different physiological activity value, but also thedanger arises that by unsuitable selection of the reduction conditionsthe aromatic six-membered ring in the hormone molecule may behydrogenated and thereby the follicle hormone action lost.

In accordance with the present invention by the application of enzymaticor phytochemical reduction methods as are summarized for example inOppenheimer, Methoden der Fermente 1929, page 1212, by Neuberg 8r Gorr,not only is the danger of hydrogenation of the aromatic nucleus avoided,but also, by reason of the asymmetrical course of the phytochemicalreduction, there is produced, in a most surprising manner, in generalonly one perfectly definite physiologically active opticalisomer.

According to the invention there is in fact obtain by the reduction ofthe follicle hormone essentially the highly active transform of thejdihydrofollicle hormone, whereas the hitherto known reduction methodsin general lead to mixtures of cisand transcompounds.

- Although the application of the phytochemical reduction methods forthe conversion of ketones into alcohols is not novel per se, yet in thepresent case its application for the production of physiologicallyactive substances with quite definite optical properties and of highestpurity constitutes a valuablephemical achievement.

The following example illustrates the inven tion:

Emample 5 166 grams of invert sugar, 600 ccs. of water In GermanyFebruary 16,

- and 50 grams of yeast are mixed. Into the mixture, which has enteredinto brisk fermentation, are allowed to drop gradually with shaking 330mg. of follicle hormone dissolved in 40 cos. of alcohol. After the wholehas been introduced, the mixture is allowed to ferment further atordinary temperature (1820) for about 40-45. hours longer and then 20grams of invert sugar, 200 cos. of water and 20 grams of yeast areintroduced. After about 90 hours the fermentation is complete, which canbe ascertained by the now only weak reduction capacity of the solutionfor Fehlings solution; the liquid is then poured off from the yeast andboth extracted several times with ether. The combined ether solutionsare evaporated the dry residue taken up with alcohol, and from thealcoholic solution any unchanged follicle hormone separated assemicarbazone'in the known manner. The residue remaining after theevaporation of the semicarbazone mother liquors is purified byrecrystallization from alcohol, if desired aftera high vacuumdistillation and in this manner the highly physiologically activedihydrofollicle hormone of M. P. 174 C. obtained.

Of course, the recovery of the reduced compounds from the fermentationmixture can be carried out in another manner from that described aboveas is evident to a chemist familiarwith the art of enzymatic reduction.Likewise many other changes and variations in the reduction conditions,the microorganisms employed and the like may be made in accordance with,the

' principles set forth herein and in the claims annexed hereto.

One may also work in the presence of known fermentation activators suchas salts, for instance primary or secondary sodium phosphate, calciumcarbonate and the like, or nitrogenous compounds that cause a more rapiddevelopment of the yeast and the like.

In a similar manner as described above, reduction products wherein thecarboxyl group is converted into a secondary alcohol group are obtainedby employing as starting material equilin 'or equilenin,oestron-benzoate or acetate or propionate or other esters,triphenylmethyl-, benzylor other ethers, or other derivatives, wherebyin 'the case of the derivatives splitting off of the substituentfrequently takes place leaving the free oestradiols.

What I claim is 1. Process for the manufacture of dihydrofolliclehormone and its unsaturated analogues dihydroequilin anddihydroequilenin, and their derivatives, comprising subjecting a memberof the group consisting of follicle hormone, equilin, eguilenin andtheir 3-derivatives to an enzymatic or phytochemical reduction until theketo group has been converted to a secondary alcohol group.

2. Process as claimed in claim 1 comprising introducing the startingmaterial into a medium wherein a reducing fermenation takes place.

3. Process as claimed in claim 1 wherein the starting material isintroduced into a. medium wherein a reducing fermentation with yeasttakes place.

4. Process as claimed in claim 1 wherein the starting material isenzymatically reduced by fermentation by means of a suspension offermenting micro-organisms in a sugar solution.

5. Process as claimed in claim 1 wherein the starting material isenzymatically reduced by fermentation of a sugar solution by means ofyeast.

6. Process as claimed in claim 1 comprising introducing the startingmaterial into a medium wherein a reducing fermentation takes place, saidmedium containing an activator capable of activating fermentation.

7. Process as claimed in claim 1 comprising introducing the startingmaterial into a medium wherein a reducing fermentation takes place, saidmedium containing an activator capable of activating fermentation andselected from the group of salts consisting of primary and secondarysodium phosphate and calcium carbonate.

8. Process for the manufacture of dihydrofollicle hormone and itsunsaturated analogues dihydroequilin and dihydroequilenin, and theirderivatives, comprising gradually introducing a solution of a member ofthe group consisting of follicle hormone, equilin, equilenin and their3- derivatives into an aqueous suspension of sugar and yeast, allowingthe mixture to ferment, thereafter extracting the mixture by means of anorganic solvent wherein the partially hydrogenated hormone is soluble,and isolating the hormone from said extract.

9. Process as claimed in claim 8 wherein unreacted starting material isremoved from the extract by converting the same into a relativelyinsoluble condensate with a ketone reagent, and

recovering the reduction product.

. WALTER SCHOEILER.

